There are some proteins which require a qualitative modification for their activation. The modification is usually carried out post-translationally (e.g. phosphorylation, processing, lipid modification) and through interactions with other proteins (e.g. binding subunits, endogenous inhibitors). Therefore, if the above regulation would be disturbed, a variety of pathological processes might be induced and cause cell death or tissue destruction. Proteases present as a modulator of intracellular signalling and capable of regulating other intracellular proteins are attractive pharmaceutical target to investigate (Trends in Biological Science, 14, 268-271 (1989)).
Calpain is an intracellular cysteine protease, the activity of which is regulated by calcium ions. It has therefore been believed that calpain functions as a regulatory molecule in cellular functions. Two calpains, .mu.-calpain and m-calpain, which require low and high micromolar Ca.sup.2+ concentration for activation, respectively, were discovered in poultry and many mammalian animals in an early stage of research. Recently, several calpains have been reported in nematodes and Drosophilas (e.g. sol and CalpA from Drosophilas and Ce-CL2 and Ce-CL3 from nematodes). Furthermore, two tissue-specific calpains also have been discovered in higher vertebrates. They are p94 (nCL-1), which is specifically expressed in the skeletal muscles of human, rat, and chicken, and nCL-2 and nCL-2', which are reported to be most highly expressed in the stomach of rats. It is known that some of modulating proteins (e.g. protein kinase C, MAP kinase) comprises a large number of isoforms to control diverse cellular functions. Therefore, it is believed that unknown calpains isoforms exist. At the present time, in humans, only three calpains are known, .mu.-calpain and m-calpain, which are ubiquitous in vertebrate tissues, and p94 (nCL-1).
Isolation of novel calpains can provide a new pharmaceutical compound and composition for treatment of diseases where normal levels of calpain are lacking, thus causing a loss of control of cell growth, i.e., diseases such as cancer. Discovery and isolation of new calpains also will contribute to a still further detailed exploration into the implication of calpain in the Ca.sup.2+ -activated signal transduction system and, should it be found to be tissue-specific, into the relationship of calpain with various tissue-specific diseases. It would also enable us to develop new drugs which would either activate or inhibit an activity of the calpain and thus be useful for the prevention and therapy of various diseases. Thus, in the technological area to which the present invention pertains, there has been a standing need for isolating novel human calpain isoforms and for developing a method for high production of such calpains.
The inventors of the present invention did much research for solving the above problems and succeeded in cloning a cDNA having a novel nucleotide sequence from a human leukocyte-derived cDNA library. They found that the protein encoded by this cDNA is a calpain. The present inventors made further investigations based on these findings, and accomplished the present invention.